Immunological control of neoplasia has been a topic of intense investigation dating back to the days of William Coley, who at the beginning of the 20th century reported potent induction of tumor remission through administration of various non-specific immune stimulatory bacterial extracts which came to be known as “Coley's Toxins” (1). Suggestions of the ability to induce anti-cancer immunological responses also came from experiments in the 1920s demonstrating that the vaccination with non-viable tumor cells mounts a specific “resistance” to secondary challenge, although at the time, the concept of MHC matching was not known and it was possible that the secondary resistance was only a product of allogeneic sensitization (2). Although the field of cancer immunotherapy has been very controversial throughout the 20th Century, with some authors actually claiming that immunological responses are necessary for tumor growth (3), the age of molecular biology has demonstrated that indeed immune responses are capable of controlling tumors from initiating, as well as in some cases inhibiting the growth of established tumors.
Originally demonstrated in the murine system, the concept of a productive anti-tumor response was associated with a cytokine profile termed Th1, whereas an ineffective anti-tumor response was associated with Th2. The prototypic method of assessing Th1 activity was by quantitation of the cytokine IFN-γ(4). At an epigenetic level it is known that the chromatin structure of Th1 and Th2 cells is distinct, thus providing a solid foundation that once a naïve T cell has differentiated into a Th1 or Th2 cell, the silenced and activated parts of the chromatin are passed to progeny cells, thus the phenotype is stable (5). Associated with such chromatin changes is the activation of the multi-gene inducing transcription factors GATA-3 (6), STAT6 (7, 8) in Th2 cells, and T-bet (9), and STAT4 (10) in Th1 cells. Accordingly studies have been performed using STAT6 knockout mice as a model of an immune response lacking Th2 influences, thus predominated by Th1. Tumors administered to STAT6 knockout animals are either spontaneously rejected (11), or immunity to them is achieved with much higher potency compared to wild-type animals (12). Furthermore immunologically mediated increased resistance to metastasis is observed (13). In agreement with the Th1/Th2 balance, mice lacking STAT4 develop accelerated tumors in a chemically-induced carcinogenesis model (14).
In the clinical situation correlation between suppressed immune responses and a higher incidence of cancer is well established. For example, natural immune deficiency such as the congenital abnormality Chediak-Higashi Syndrome, in which patients have abnormal natural killer cell function, is associated with an overall weakened immune response. In this population, the overall incidence of malignant tumors is 200-300 times greater than that in the general population (15). In another example, a specific polymorphism of the IL-4 receptor gene that is known to be associated with augmented Th2 responses was investigated in an epidemiological study. Multivariate regression analysis showed that the specific genotype of the IL-4R associated with augmented Th2 activity was an independent prognostic factor for shorter cancer survival and more advanced histopathological grade (16). In addition to inborn genetic abnormalities, the immune suppressive regimens used for post-transplant antirejection effect are associated with a selective inhibition of Th1 responses (17-19). In support of the concept that suppression of Th1 immunity is associated with cancer onset, the incidence of cancer in the post-transplant population is markedly increased in comparison to controls living under similar environmental conditions (20-25). In terms of disease associated immune suppression, HIV infected patients also have a marked predisposition to a variety of tumors, especially, but not limited to lymphomas, as a result of immunodeficiency (26).
Although the above examples support a relation between immune suppression (or Th2 deviation) and cancer, the opposite situation, of immune stimulation resulting in anticancer response, is also documented. Numerous clinical trials using antigen specific approaches such as vaccination with either tumor antigens alone (27, 28), tumor antigens bound to immunogens (29, 30), tumor antigens delivered alone (31) or in combination with costimulatory molecules by viral methods (32), tumor antigens loaded on dendritic cells ex vivo (33-35), or administration of in vitro generated tumor-reactive T cells (36), have all demonstrated some clinical effects. Unfortunately, to date, there is no safe, reproducible, and mass-applicable method of therapeutically inducing regression of established tumors, or metastasis via immunotherapy. Approved immunotherapeutic agents such as systemic cytokine administration are associated with serious adverse effects, as well as mediocre responses and applicability to a very limited patient subset.
Accordingly, there is a need in the art to develop successful immunotherapy capable of stimulating specific immune responses that only target neoplastic tissue, or components of the host tissue whose activity is necessary for the progression of neoplasia (ie endothelium). The development of such a successful immunotherapy is hindered by suppression of the host immune system by the cancer. Experiments in the 1970s demonstrated the existence of immunological “blocking factors” that antigen-specifically inhibited lymphocyte responses. Some of this early work involved culturing autologous lymphocytes with autologous tumor cells in the presence of third party healthy serum. This culture resulted in an inhibition of growth of the autologous tumor as a result of the lymphocytes. Third party lymphocytes did not inhibit the growth of the tumor. Interestingly when autologous serum was added to the cultures the lymphocyte mediated inhibition of tumor growth was not observed. These experiments gave rise to the concept of antigen-specific “blocking factors” found in the body of cancer patients that incapacitate successful tumor immunity (37-39).
More recent demonstration of tumor-suppression of immune function was seen in experiments showing that T cell function is suppressed in terms of inability to secrete interferon gamma due to a cleavage of the critical T cell receptor transduction component, the TCR-zeta chain. Originally, zeta chain cleavage was identified in T cells prone to undergo apoptosis. Although a wide variety of explanations have been put forth for the cleavage of the zeta chain, one particular cause was postulated to be tumor-secreted microvesicles.
Microvesicles secreted by tumor cells have been known since the early 1980s. They were estimated to be between 50-200 nanometers in diameter and associated with a variety of immune inhibitory effects. Specifically, it was demonstrated that such microvesicles could not only induce T cell apoptosis, but also block various aspects of T cell signaling, proliferation, cytokine production, and cytotoxicity. Although much interest arose in said microvesicles, little therapeutic applications developed since they were uncharacterized at a molecular level.
Research occurring independently identified another type of microvesicular-like structures, which were termed “exosomes”. Originally defined as small (i.e., 80-200 nanometers in diameter), exosomes were observed initially in maturing reticulocytes. Subsequently it was discovered that exosomes are a potent method of dendritic cell communication with other antigen presenting cells. Exosomes secreted by dendritic cells were observed to contain extremely high levels of MHC I, MHC II, costimulatory molecules, and various adhesion molecules. In addition, dendritic cell exosomes contain antigens that said dendritic cell had previously engulfed. The ability of exosomes to act as “mini-antigen presenting cells” has stimulated cancer researchers to pulse dendritic cells with tumor antigens, collect exosomes secreted by the tumor antigen-pulsed dendritic cell, and use these exosomes for immunotherapy. Such exosomes were seen to be capable of eradicating established tumors when administered in various murine models. The ability of dendritic exosomes to potently prime the immune system brought about the question if exosomes may also possess a tolerance inducing or immune suppressive role. Since it is established that the exosome has a high concentration of tumor antigens, the question arose if whether exosomes may induce an abortive T cell activation process leading to anergy. Specifically, it is known that numerous tumor cells express the T cell apoptosis inducing molecule Fas ligand.
Fas ligand is an integral type II membrane protein belonging to the TNF family whose expression is observed in a variety of tissues and cells such as activated lymphocytes and the anterior chamber in the eye. Fas ligand induces apoptotic cell death in various types of cells target cells via its corresponding receptor, CD95/APO1. Fas ligand not only plays important roles in the homeostasis of activated lymphocytes, but it has also been implicated in establishing immune-privileged status in the testis and eye, as well as a mechanisms by which tumors escape immune mediated killing. Accordingly, given the expression of Fas ligand on a variety of tumors, we and others have sought, and successful demonstrated that Fas ligand is expressed on exosomes secreted by tumor cells (40).
Due to the ability of exosomes to mediate a variety of immunological signals, the model system was proposed that at the beginning of the neoplastic process, tumor secreted exosomes selectively induce antigen-specific T cell apoptosis, through activating the T cell receptor, which in turn upregulates expression of Fas on the T cell, subsequently, the Fas ligand molecule on the exosome induces apoptosis. This process may be occurring by a direct interaction between the tumor exosome and the T cell, or it may be occurring indirectly by tumor exosomes binding dendritic cells, then subsequently when T cells bind dendritic cells in lymphatic areas, the exosome actually is bound by the dendritic cell and uses dendritic cell adhesion/costimulatory molecules to form a stable interaction with the T cell and induce apoptosis. In the context of more advanced cancer patients, where exosomes reach higher concentrations systemically, the induction of T cell apoptosis occurs in an antigen-nonspecific, but Fas ligand, MHC I-dependent manner.
The recent recognition that tumor secreted exosomes are identical to the tumor secreted microvesicles described in the 1980s (41), has stimulated a wide variety of research into the immune suppressive ability of said microvesicles. Specifically, immune suppressive microvesicles were identified not only in cancer patients (42, 43), but also in pregnancy (44-46), transplant tolerance (47, 48), and oral tolerance (49, 50) situations.
Previous methods of inducing anti-cancer immunity have focused on stimulation of either innate or specific immune responses, however relatively little work has been performed clinically in terms of de-repressing the immune functions of cancer patients. Specifically, a cancer patient having tolerance-inducing exosomes has little chance of mounting a successful anti-tumor immune response. This may be one of the causes for mediocre, if not outright poor, results of current day immunotherapy.
Others have attempted to de-repress the immune system of cancer patients using extracorporeal removal of “blocking factors”. Specifically, Lentz in U.S. Pat. No. 4,708,713 describes an extracorporeal method of removing proteins approximately 200 kDa, which are associated with immune suppression. Although Lentz has generated very promising results using this approach, the approach is: a) not-selective for specific inhibitors; b) theoretically would result in loss of immune stimulatory cytokines; c) is not applicable on a wide scale; and d) would have no effect against tumor-secreted microvesicles which are much larger than 200 kDa.
The recently discovered properties of microvesicles in general, and tumor microvesicles specifically, have made them a very promising target for extracorporeal removal. Properties such as upregulated expression of MHC I, Fas ligand, increased affinity towards lectins, and modified sphingomyelin content allow for use of extracorporeal devices to achieve their selective removal. Additionally, the size of microvesicles would allow for non-selective removal either alone or as one of a series of steps in selective removal.